CN112941204B - Broiler abdominal fat rate molecular marker LPIN1g.256 and detection method and application thereof - Google Patents

Broiler abdominal fat rate molecular marker LPIN1g.256 and detection method and application thereof Download PDF

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CN112941204B
CN112941204B CN202110314086.5A CN202110314086A CN112941204B CN 112941204 B CN112941204 B CN 112941204B CN 202110314086 A CN202110314086 A CN 202110314086A CN 112941204 B CN112941204 B CN 112941204B
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罗庆斌
张细权
聂庆华
张德祥
罗文�
李恒丰
李红梅
叶茂
詹惠娜
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Abstract

The invention discloses a broiler abdominal fat rate molecular marker LPIN1g.256 and a detection method and application thereof, and relates to the technical field of poultry genetic breeding. The invention provides SNP related to broiler abdominal fat percentage, and the sequence of the SNP is shown as SEQ ID NO: 1. The invention also provides a specific primer group for detecting the SNP and a specific detection method, which can detect the SNP sequence efficiently, sensitively and quickly so as to screen and identify the broiler varieties with low abdominal fat percentage. The implementation of the invention can effectively screen the broiler chickens with low abdominal fat percentage, provides a new technical scheme for the genetic breeding of the broiler chickens, and is suitable for large-scale popularization and application in the broiler chicken breeding industry.

Description

Broiler abdominal fat rate molecular marker LPIN1g.256 and detection method and application thereof
Technical Field
The invention relates to the technical field of poultry genetic breeding, in particular to a broiler abdominal fat percentage molecular marker LPIN1g.256 and a detection method and application thereof.
Background
The sources of unique flavors in chicken are mainly intramuscular fat and subcutaneous fat. On one hand, tenderness and juiciness in the meat quality index are caused by the dissolution of intramuscular fat to muscle fiber; on the other hand, in the process of degrading the lipid of the intramuscular fat and the subcutaneous fat of the chicken, the fragrance generated by the abundant phospholipid is an important part for forming the flavor of the chicken, meanwhile, the unsaturated fatty acid in the muscle is oxidized when being heated, and the oxidation product is further decomposed into volatile substances such as olefine aldehyde and the like, and is an important component for forming the fragrance of the chicken. And the unique flavors of various famous local breeders in China, such as Beijing fatty chicken, Qingyuan pockmarked chicken, Pengxian yellow chicken and the like, are all derived from subcutaneous fat and intramuscular fat deposition to a certain degree. However, the method is limited by the fat deposition rule in chicken bodies, and the problem that the excessive fat deposition of the broilers is regarded as a problem which is considerably important by breeding workers in the genetic breeding of the broilers and an important way for improving the productivity of yellow-feathered broilers is solved.
Molecular Marker Assisted Selection (MAS) is a commonly used molecular breeding technique. MAS can be directly analyzed for polymorphisms of genetic material, such as Single Nucleotide Polymorphisms (SNPs). The molecular marker assisted selection technology has a quite long history in breeding of other livestock, compared with the traditional breeding method, MAS has the advantages of wide existence range, stable heredity, intuition, accuracy and the like, and is not applied to breeding of chickens on a large scale at present. On one hand, the molecular marker for the economic traits of the chicken is less researched and excavated, and the molecular marker for selecting important economic traits can be applied to actual breeding; on the other hand, the detection of the molecular marker is often higher in cost, and the cost of applying the molecular marker assisted selection technology in chicken breeding with short generation intervals and large breeding groups is higher than that of the conventional breeding method. However, with the development of more low-cost detection technologies and the development of molecular markers, the application cost of the molecular marker assisted selection technology in chicken breeding is remarkably reduced, so that candidate genes related to important economic traits of chickens on the molecular level are further mined, and the breeding work of high-quality chickens can be effectively promoted by researching the polymorphism of the candidate genes and establishing related molecular genetic markers.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a broiler abdominal fat rate molecular marker LPIN1g.256 and a detection method and application thereof. According to the known sequence of the LPIN1 gene, an SNP locus associated with the abdominal fat rate of yellow-feathered broilers is searched by utilizing direct sequencing, and the correlation analysis is carried out on the SNP typing result and the group character through the flight time mass spectrum analysis, so that a molecular marker associated with the abdominal fat rate of yellow-feathered broilers is obtained, and a new molecular marker resource is provided for the marker-assisted selection of chickens.
In order to realize the purpose, the following scheme is provided:
in the first aspect, a molecular marker LPIN1 g.256C > G related to the abdominal fat percentage of broilers is provided, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and the base 256 position of the sequence is C or G (namely, the base C is replaced with the base G).
In a second aspect, the application of the molecular marker or the reagent for detecting the molecular marker in screening the broiler chickens with low abdominal fat percentage is provided.
In some embodiments, the reagent for detecting the molecular marker comprises a specific primer set for detecting the molecular marker. In some embodiments, the nucleotide sequence of the specific primer set is shown in SEQ ID NO.2-3
In a third aspect, the invention provides a method for screening broiler chickens with low abdominal fat percentage by using molecular marker LPIN1 g.256C > G, which comprises the following steps:
(a) extracting DNA of the chicken to be detected;
(b) obtaining and sequencing a target fragment: amplifying the DNA of the chicken to be detected by PCR by using a primer group capable of amplifying the molecular marker sequence, sequencing the obtained product, and analyzing the sequence base;
(c) according to the analysis results, individuals with low abdominal fat percentage are selected during breeding.
In some embodiments, the nucleotide sequences of the primer sets are set forth in SEQ ID nos. 2-3, and individuals of the GG or CC genotype are retained when bred.
In some embodiments, the reaction conditions for the PCR amplification in step (b) are: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 25s, annealing at 58 ℃ for 25s, and extension at 72 ℃ for 10s, for 32 cycles; extension at 72 ℃ for 5 min.
In some embodiments, the reaction system for PCR amplification in step (b) comprises: DNA template 100ng, 2X M5 HiPerplus Taq HiFi PCR mix 15. mu.l, upstream and downstream primers 0.4. mu.l each, ddH 2 Make up to 30. mu.l of O.
The invention discloses the following technical effects:
the SNP locus selected by the invention is unique, has very high generalized heritability, and can accurately screen out the broiler chickens with low abdominal fat percentage. The screening method is simple and convenient to operate, high in speed, low in cost and high in accuracy. The implementation of the invention can effectively screen the broiler chickens with low abdominal fat percentage, provides a new technical scheme for the genetic breeding of the broiler chickens, and is suitable for large-scale popularization and application in the broiler chicken breeding industry.
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FIG. 1 shows the results of agarose gel electrophoresis. Wherein M is a Marker of DL2000 (2000, 1000, 750, 500, 250, 100bp from top to bottom respectively); 1. 2, 3, 4, 5 and 6 are PCR products;
FIG. 2 shows the different genotypes of the LPIN1 g.256C > G sites.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are all commercially available reagents and materials unless otherwise specified.
Example 1SNP site screening
1. Test object
Two samples of a high abdominal fat rate group (marked as a group H) and a low abdominal fat rate group (marked as a group L) are selected from five varieties by taking the abdominal fat rate as a selection standard, and the selected varieties and the number of the two samples in each variety are as follows: 12 Guangxi Lingshan chickens, 12 short-footed yellow chickens, 12 Jiangfeng Ma yellow chickens, 10 Zhicheng apricot flower chickens and 10 recessive white feather cocks.
Statistics of measurement data of traits such as abdominal fat percentage of two tail samples of each variety are shown in Table 1. In two tail samples of Guangxi Lingshan, there was no significant difference in live weight between the H group and the L group (P >0.05), the average abdominal fat weight of the H group (173.25g) was about 4.61 times higher than the average abdominal fat weight of the L group (37.58g), and the average abdominal fat rate of the H group (9.31%) was about 3.54 times higher than the average abdominal fat rate of the L group (2.63%).
TABLE 1 two-tailed information for screening SNP sites
Figure BDA0002991063380000031
Figure BDA0002991063380000041
2. Selection of candidate genes
Candidate gene LPIN1 associated with fat deposition in slow yellow broiler was selected from prior studies.
3. Extraction of genomic DNA
Extracting chicken genome DNA by using a blood DNA extraction kit, extracting the genome DNA of a blood sample according to the operation of a blood DNA extraction instruction, detecting the concentration and OD value of the DNA sample, and enabling the DNA concentration to be more than 25 ng/mu l and the OD value to be larger than 260 /OD 280 Samples with a ratio between 1.7 and 1.8 were stored at-20 ℃ until use.
4. Candidate gene primer
According to the candidate genome sequence of chicken published in NCBI, primers are designed and amplified by using an NCBI online primer design tool (https:// www.ncbi.nlm.nih.gov/tools/primer-blast), and a gene fragment with the size of about 1000bp is amplified from the 3' end, wherein the specific primers are shown in Table 2:
TABLE 2 PCR amplification primers for candidate genes
Figure BDA0002991063380000042
5. PCR amplification and sequence analysis of candidate gene fragments
The PCR reaction system is as follows:DNA template 100ng, 2X M5 HiPer plus Taq HiFi PCR mix 15. mu.l, upstream and downstream primers 0.4. mu.l each, ddH 2 Make up to 30. mu.l of O.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 25s, annealing at 58 ℃ for 25s, and extension at 72 ℃ for 10s, for 32 cycles; extending for 5min at 72 ℃; storing at 12 deg.C.
Carrying out agarose gel electrophoresis on the amplification product, and judging the result according to the size of the product:
and (3) detecting the PCR amplification product by using EB-containing 1.5% agarose gel electrophoresis under the voltage of 100V for 20min, observing a band (about 1000bp, shown in figure 1) in a gel imaging system, recovering and purifying, finally sequencing, analyzing sequence bases, and screening candidate gene SNP sites (a partial sequencing peak is shown in figure 2).
Example 2 time-of-flight mass spectrometry
A flight time mass spectrum typing technology of Beijing Liuhe Huada Dageno science and technology Limited company is adopted to perform genotyping on candidate SNP sites obtained by direct sequencing in 500N 409 (Guangxi Lingshan chickens) groups, and the combination of SNP typing results and abdominal fat related slaughter traits (abdominal fat rate and abdominal fat weight) is subjected to correlation analysis through SPSS 21.0 software, so that SNP obviously related to the abdominal fat rate of yellow-feathered broilers is identified, and is shown in the following table 3.
TABLE 3SNPs information Table
Figure BDA0002991063380000051
Example 3 analysis of the differences between genotypes of SNPs significantly associated with abdominal fat Rate
The results of analysis of different genotypes at the SNP sites significantly correlated with abdominal fat percentage, abdominal fat weight, subcutaneous fat thickness, and other traits are shown in table 4 by SPSS 21.0 software.
TABLE 4 genotype differential analysis of SNPs significantly associated with abdominal fat percentage
Figure BDA0002991063380000052
Figure BDA0002991063380000061
Note: the number in parentheses after the genotype indicates the number of samples of the genotype; the same letter between 3 genotypes means that the difference is not significant (P >0.05), different letters means that the difference is significant (P <0.05), and no letter mark means that the difference between the genotype and other 2 genotypes is not significant.
Therefore, the broiler chickens with low abdominal fat percentage can be selected in broiler breeding through LPIN1 gene marker-assisted selection, and breeding work of high-quality chickens is promoted.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> southern China university of agriculture
<120> broiler abdominal fat rate molecular marker LPIN1g, 256 and detection method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 327
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> mutation
<222> (256)..(256)
<223> s = c or g
<400> 1
aattagcagg caaacgcgat cactgggaaa ttatattctc tggagctttg ttgtcaacag 60
ttaaaacctg aaggcgacat ttttgtatat ggccgaaacc aaggctagtt ttagtagcct 120
gtgcaaagaa tgggggaaaa ggaccacttc aagatgtcgt ctgaggatga agagcagcct 180
gagagccctc ctggctcccc ttggtcttgg gtaaggtctt atgccttaag gttgcataaa 240
tagcatttta aagctstagc agagactgtg ctgtatgaac ctggatagaa ctgaatgaaa 300
ccagctttct taatctgaat aaaacca 327
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gctctggttt acggagctca t 21
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcactgtacc cagaccactc 20

Claims (7)

1. The application of a molecular marker related to the abdominal fat percentage of broiler chickens or a reagent for detecting the molecular marker in screening of broiler chickens with low abdominal fat percentage is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, wherein the 256 th base of the sequence is an SNP (single nucleotide polymorphism) site, and the base is C/G.
2. The use according to claim 1, wherein the reagent for detecting the molecular marker of claim 1 comprises a specific primer set for detecting the molecular marker of claim 1.
3. The use according to claim 1, wherein the reagent for detecting the molecular marker of claim 1 is a primer, and the nucleotide sequence of the primer is shown in SEQ ID No. 2-3.
4. A method for screening broiler chickens with low abdominal fat percentage by using the molecular marker in claim 1 is characterized by comprising the following steps:
(a) extracting DNA of the chicken to be detected;
(b) obtaining and sequencing a target fragment: amplifying the DNA of the chicken to be detected by PCR by using a primer group capable of amplifying the molecular marker sequence in claim 1, sequencing the obtained product, and analyzing the sequence base;
(c) according to the analysis result, individuals retaining GG or CC genotypes are selected during breeding.
5. The method of claim 4, wherein the nucleotide sequence of the primer set is shown in SEQ ID NO. 2-3.
6. The method of claim 4, wherein the reaction conditions for the PCR amplification in step (b) are: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 25s, annealing at 58 ℃ for 25s, and extension at 72 ℃ for 10s, for 32 cycles; extension at 72 ℃ for 5 min.
7. The method of claim 4, wherein the reaction system for PCR amplification in step (b) comprises: DNA template 100ng, 2X M5 HiPerplus Taq HiFi PCR mix 15. mu.l, upstream and downstream primers 0.4. mu.l each, ddH 2 Make up to 30. mu.l of O.
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